Ion channel glutamate receptors are ligand-gated transmembrane proteins that can be activated by the binding of glutamate, the principal excitatory neurotransmitter in the brain. Ionotropic glutamate receptors (iGluRs) are, therefore, the major excitatory neurotransmitter receptor proteins in the mammalian brain. As such, these receptors play special roles in brain activities, such as memory and learning, and have been implicated in a variety of neurological diseases, such as post-stroke cellular lesion and amyotrophic lateral sclerosis (1, 2).
When glutamate is released from a presynaptic neuron and binds to a postsynaptic glutamate receptor, the receptor rapidly changes its conformation and transiently forms an open ion channel, thus resulting in a change of the postsynaptic membrane potential. A postsynaptic potential of sufficient strength triggers an action potential, which will in turn propagate the initial nerve impulse. The major function of iGluRs is to mediate fast synaptic neurotransmission underlying the basic activities of the brain, for example, memory and learning. Excessive activation of ionotropic glutamate receptors, particularly the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) subtype, is known to induce calcium-dependent excitotoxicity. Excitotoxicity has been considered as a general mechanism underlying a number of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), stroke, Alzheimer's disease and Parkinson's syndrome.
AMPA receptors are one of the three subtypes of glutamate ion channels that also include kainate and N-methyl-D-aspartate (NMDA) subtypes (1, 3-5). AMPA receptors mediate most of fast synaptic neurotransmission in the mammalian central nervous system, and their function and regulation are critical for synaptic plasticity (5, 6). GluA1-4 (previously known as GluR1-4 or GluRA-D) encode four subunits of mammalian AMPA receptors. The primary molecular architecture of AMPA receptor subunits is most likely similar, given the fact that all subunits have ˜900 amino acids and share 70% homology of the encoding genes, although the genes are alternatively spliced and edited (1, 3, 4). AMPA receptor subunits are differentially expressed and developmentally regulated. For instance, in embryonic rat brain, GluA2 mRNA is ubiquitous (7). GluA1-3 are expressed in greater proportion in regions such as hippocampus (8), whereas GluA4 is mainly expressed early during development (9, 10). Although GluA1-4 can form homomeric channels individually (11, 12), each subunit has some distinct functional properties. For example, in response to the binding of glutamate, each of the GluA2-4 homomeric receptors opens the channel, with a kinetic rate constant about several-fold larger than GluA1 does, yet all AMPA receptors close their channels with roughly a similar rate (13, 14).
Using inhibitors to dampen the excessive activity of these receptors may serve as a treatment for neurological disorders such as amyotrophic lateral sclerosis (ALS) or Huntington's disease. To date, Riluzole, an inhibitor of presynaptic glutamate release, is the only drug to produce a significant benefit to the survival of ALS patients. The number of glutamate receptor inhibitors currently available is limited and these inhibitors generally show cross activity to other receptors, for example, kainate receptors. The cross activity is not desirable, because the AMPA and kainate receptors have functional differences. Furthermore, the majority of AMPA receptor inhibitors have poor water solubility. In addition, there is a lack of an assay of inhibitor-receptor interactions within the microsecond (μs) to millisecond (ms) time domain. This is because an AMPA receptor opens its channel in the μs time scale and desensitizes within a few ms in the continued presence of glutamate. Consequently, the affinity of all AMPA receptor inhibitors has been determined only with the desensitized receptors. These deficiencies have significantly hampered drug development.
Because proteins are generally dynamic and adapt a specific conformation for function, using molecular agents that bind selectively to a specific protein conformation among its conformational repertoire is thus a powerful means to exert a tighter molecular recognition to more effectively regulate the existing function of that protein, and to even engineer a new protein function. For instance, small chemical compounds have been found to stabilize a conformation for some apoptotic procaspases to induce autoproteolytic activation of these proenzymes. Catalytic antibodies have been created, based on transition-state structural analogs, to accelerate chemical reactions by stabilizing their rate-determining transition states along reaction pathways. Developing inhibitors to control excessive receptor activity has been a long pursued therapeutic strategy for a potential treatment of these neurological disorders and diseases.
Additionally, developing inhibitors to selectively target a single subunit of a multi-subunit protein or receptor family is a worthy effort for the following reasons. First, the role of the single subunit can be uniquely tested in a complex biological background, such as in vivo, leaving other subunits untouched. Such a test can be carried out at any particular time if the target function changes during development. In this scenario, the function of this subunit can be inhibited in a reversible, graded fashion in that the degree of inhibition of the protein function can be manipulated by the amount and the time of exposure when the inhibitor is applied, and such an inhibition can be reversibly relieved when the inhibitor is removed. Second, if the inhibitor is a drug candidate, selectivity is generally a desired property. A drug with higher selectivity may have a higher therapeutic effect when the excessive activity of a single protein subunit to which the drug molecule binds is linked to the pathogenesis of a disease. Third, development of an inhibitor to exclusively differentiate its binding to and inhibition of one subunit can provide valuable insights into the structural and functional differences of the subunit from all other subunits of the same family. As such, the most effective way to probe the structure of a particular subunit and to regulate the function of that subunit may be found.
Given the similarities and differences among various AMPA receptor subunits, it would be useful to develop subunit-selective inhibitors of AMPA receptors. What is needed, therefore, are subunit-selective AMPA glutamate receptor inhibitors that are characterized by a high affinity for its target, preferably in the nanomolar range, specificity targeting a single subunit of a glutamate receptor, excellent water solubility and relevance of its inhibitory properties to the functional forms of the receptor rather than the desensitized receptor forms.